DS11 (KCTC 0231BP), novel Bacillus sp. strain and novel phytase produced by it

ABSTRACT

The strain Bacillus sp. DS11 (KCTC 0231BP) is disclosed and a phytase produced by DS11 having the following characteristics: optimum temperature: 65° C.; optimum pH: 7.0; molecular weight: 43,000 dalton; isoelectric point: 5.6; and a specified N-terminal amino acid sequence. The bacterial strain DS11 or the phytase it produces can be used as an animal feed additive.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel phytase produced from novel strainBacillus sp. DS11 (KCTC 0231BP) and more precisely, to a novel strainBacillus sp. DS11 and phytase enzyme enhancing the phosphatebioavailability present in grains supplied to monogastric animals.

2. Description of the Prior Art

Phytase is an enzyme to degrade phytic acid into phosphate and phosphateinositol. 50 to 70% of phosphate in grain used as livestock feeds existsin form of phytic acid but phytase is not present in monogastric animalssuch as hens and hogs, resulting in low phosphate availibility.

Therefore undigested phytic acid(phytictaine) released to a water sourcebecame one of the serious environment contamination sources causing toeutrophication in small lakes or tides. With above consideration,because monogastric animals can't utilize phytic acid in their intestinephytic acid chelates to water due to chelation with a trace amount ofminerals, amino acids and vitamins which are very important to themetabolism of livestock. These formed water-insoluble, undigestablechelate complexes released to feces change the environmental ecosystemto induce a serious environmental pollution.

In view of these situations, the application of phytase into thelivestock feeds will reduce the supply of inorganic phosphate due toincrease of phosphate bioavailibility in livestock, leading to economicbenefits, and improving the availibility of phosphate, and otherbioactive substances, leading to reduction of the environmentalcontamination.

By these reasons, the utilization of phytase in livestock is veryimportant. A law regulating the amount of phosphate in animal waste wasestablished in 1996 in Korea and in European countries it is alreadymandatory to add phytase in the feeds of animals.

The addition of phytase in the feeds may greatly improve theproductivity of livestock by enhancing the availability of somebioactive substances (phosphate, calcium and zinc etc.) which, otherwisechelate with phytictaine and lose their activity.

As the result, the use of feeds containing phytase in livestock canenhance the availibility of feeds and reduce the environmentalcontamination caused by phosphate.

For the aforementioned benefits, intensive studies about phytaseincluding the effects of phytase on animals Young et al., Addition ofmicrobial phytase to diet of young pigs, J Anim Sci 71(8)2147-50 (1993);Lei et al., Calcium level affects the efficacy of supplemental microbialphytase in corn-soybean meal diets of weaning pigs, J Anim Sci72(1)139-43 (1994); and Mroz et al., Apparent digestibility andretention of nutrients bound to phytate complexes as influenced bymicrobial phytase and feeding regimen in pigs, J Anim Sci 27(1):126-32(1994); Z. Mroz et al., 1994) have been performed mainly in EuropeUllah., A. H., The complete primary structure elucidation ofasperigillus ficuum (niger), pH 2.5-optimum acid phosphatase by Edmandegradation, Biochem Biophy Res Commun 203(1):182-89 (1994); Ehrlich, etal., Identification and cloning of a second phytase gene (phyB) fromAspergillus niger (ficuum); and Piddington et aL, The Cloning andsequencing of the genes encoding phytase (phy) and pH 2.5-optimum acidphosphatase (aph) from Asperigillus niger var. awamori, Gene133(1):55-62 (1993).However, since phytase can cleave only a limitednumber of phosphates and it mostly produced by molds which have longgrowing period, it is uneconomical in terms of mass production. Inaddition, it is difficult to use the phytic acid as an additive formonogastric animals since it is unsuitable to their physiologicalmechanism.

SUMMARY OF THE INVENTION

Therefore, the inventor et al. have identified a novel phytase-producingmicroorganism among hundred kinds of molds, Actinomycetes, bacteria,etc., obtained from soils and barns throughout the country, in an effortto produce phytase having excellent enzymatic potency and shorten theproduction period compared with the conventional phytase. Because of thehigh enzymatic potency of this novel microorganism and its physiologicalsuitability to use for monogastric animals and short period ofkproduction compared to those of conventional enzymes the inventor etal. have judged that this enzyme has novelty and they have completedthis invention.

The object of this invention is to provide novel strain Bacillus sp.DS11 (KCTC 0231BP) and phytase enzyme, which is suitable to use formonogastric animals with excellent properties and more shortenedproduction period.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is electron microscope photograph (×10,000) of Bacillus sp. DS11(KCTC 0231BP) strain, a novel strain according to this invention,

FIG. 2 is a SDS-PAGE electrophoresis analysis of novel phytase producedby Bacillus sp. DS11 (KCTC 0231BP), A: precipitation of acetone, B:resource S, C: superose 12 HR 10/30

FIG. 3 is a graph measuring of novel phytase produced by Bacillus sp.DS11 (KCTC 0231BP), a novel strain, in accordance with EXPERIMENTALEXAMPLE 2 of this invention.

DETAILED DESCRIPTION OF THIS INVENTION

This invention relates to a novel phytase produced from novel strainBacillus sp. DS11 (KCTC 0231BP) and particularly, to a novel strainBacillus sp. DS11 and phytase enzyme enhancing the phosphatebioavailability present in grains supplied to monogastric animals. Thenovel strain Bacillus sp. DS11 (KCTC 0231BP) was deposited with theKorea Research Institute of Bioscience and Biotechnology KoreanCollection for type cultures, Korean Patent Strain Deposit Associates,located in #52, Oun-dong, Yusong-ku Taejeon 305,333, Republic of Korea,on Feb. 1, 1996, under Deposit Accession Number KCTC0231BP.

This invention is described in detail as set forth hereunder:

This invention relates to novel strain Bacillus sp. DS11 (KCTC 0231BP).

Further, this invention includes its novel enzyme phytase whoseN-terminal amino acid sequence is SEQ. ID NO: 1 under the followingconditions:

Optimal temperature: 65° C.

Optimal pH: 7.0

Molecular weight: 43,000 dalton

Isoelectric point: 5.6

In addition, this invention includes a method to use said microorganismas feed additives.

This invention is described in more detail as set forth hereunder:

This invention relates to a novel strain Bacillus sp. DS11 (KCTC 0231BP)and novel phytase produced from said strain. The procedure for isolatingand identifying said novel microorganism is as follows:

[Isolation of Novel Microorganisms]

From several thousands of strains obtained from soils and in barnsthroughout the country, a strain was isolated which has excellentresolution in phytase screen plate containing 15 g of D-glucose, 5 g ofcalcium phytate, 5 g of NH₄NO₃, 0.5 g of MgSO₄·7H₂O, 0.5 g of KCl, 0.01g of FeSO₄·7H₂O, 0.01 g MnSO₄·4H₂O and agar 15 g at pH 7.0/l.

[Identification of Novel Microorganism]

The morphological property of the strain isolated from the aboveprocedure is as follows:

1) Morphological Property

In the grams staining test of the strain cultivated in an optimal growthmedium, the strain was proven to be gram-positive. FIG. 1, the pictureof electron microscope shows that strain is a rod type of 0.8˜1.8 μm incell size.

In the growth test of the thermally inactivated cells at 80° C., saidstrain has formed thermostable spores. Further, its catalase test inwhich it shows the positive response demonstrates that it coincides withthe morphological property of Bacillus sp.

The physiological property of said strain is as follows:

2) Physiological Property

The test results of physiological property of the strain are representedin the following table 1. As shown in the table 1, it is assumed thatstrain is a facultative microorganism which may be grow in both aerobicand anaerobic states. With the difference of grown strains found at 50°C. and pH 5.7 compared with Bacillus pantothenticus, the strain of thisinvention is a mutant derived from Bacillus pantothenticus.

TABLE 1 Bacillus Strain of Property pantotheniticus this inventionCatalase + + Anaerobic culture + + V-p. test − + Acid-formingglucose + + L-arabinose − + D-xylose − − D-mannitol − + HydrolysisCasein d + Gelatin + + Starch + + Synthesis Indole − − Dihydroacetone −− Growth pH 6.8 + + 5.7 − + Concentration of  2% + + growth salt  5% + +(at NaCl)  7% + + 10% + + Growth temperature  5° C. − − 10° C. − − 30°C. + + 40° C. + + 50° C. − + Note) +: positive. −: negative. d:different from species.

The chemical property of the strain obtained as above is as follows:

3) The Chemical Property of Strain

After harvesting the strain, the test results of their variousproperties (e.g., G+C content, fatty acid composition, Murein Type andmain melaquinone) are represented in the following table 2.

As shown in the above table 2, it is noted that the strain of thisinvention is similar to Bacillus pantotheniticus in terms of G+Ccontent, murein type and main melaquinone including fatty acid of strainbut it seems to difficult to make a judgement that both strains are thesame species. Thus, it seems that the strain of this invention is amutant derived from Bacillus pantothenticus.

TABLE 2 Bacillus Strain of Property pantotheniticus this invention G + Ccontent 36.9 45.6 Murein type Mezo-DAP Mezo-DAP Main melaquinone MK-7MK-7 Fatty acid of strain (%) 14:0 ISO 4.71 1.96 14:0 1.50 1.21 15:0 ISO19.34 18.72 15:0 ANTEISO 37.95 38.51 16:0 ISO 10.01 6.16 16:0 9.77 9.7717:0 ISO 4.37 9.02 17:0 ANTEISO 12.00 11.20 18:0 x 2.03

By compiling above mentioned morphological, physiological and chemicalproperties, it is revealed that the strain of this invention belongs toBacillus sp. from Sneath, P., Endospore-forming Gram-Positive Rods andCocci, Bergey's Manual of Systematic Bacteriology 2:1104-1139 (1984).Therefore, the microorganism isolated was nominated as Bacillus sp. DS11and deposited on Feb. 1, 1996, to Korea Research Institute of Bioscienceand Biotechnology Korean Collection for type cultures, Korean PatentStrain Deposit Associations located in #52, Oun-dong, Yusong-ku, Taejeon305-333, Republic of Korea, with an accession number KCTC 0231BP.

This invention is described in more detail by the following examples,but the claims are not limited to these examples.

EXAMPLE 1

Production of Novel Phytase

To produce phytase, a medium containing 6% wheat bran, NH₂NO₃ 0.04%,0.02% MgSO₄·7H₂O, 1.0% casein hydrolysate, 0.05% KH₂PO₄, 0.04% K₂HPO₄and 0.2% CaCl₂, was adjusted to pH 6.5 and sterilized at 121° C. for 15minutes.

Then, with the same medium composition, 1% seed culture cultivated in aflask at 37° C. for 12 hours was inoculated into the medium in order toproduce the enzyme.

The potency of produced enzyme was measured as follows: By using asubstrate, comprising 2 mM phytic acid sodium salts and 2 mM CaCl₂ in0.1 M Tris buffer solution (pH 7.0), the enzyme was reacted at 37° C.for 30 minutes to measure the amount of phosphoric acid generated. Oneunit of the enzymatic potency is equivalent to the enzymatic amountdegrading 1 μ mol of inorganic phosphate per 1 minute. As a result ofsaid measurement, the novel enzyme has the enzymatic potency of 0.3 unitper protein mg. The maximum amount of enzyme in fermentation yielding0.6 unit/mg was obtained when 30 l of strains, a working volume, wascharged to a 50 l fermenter and cultivated at 37° C. for 48 hours underthe following conditions: air influx—0.8 vvm; stirring rate—150 rpm.Compared with the enzymatic production amount of 0.3 unit/mg whenphytase enzyme derived from Aspergillus ficuum is cultivated for 96hours [Gibson, D. M., Some modification to the media for rapid automateddetection of salmonellas conductance measurement, J Appl Bacteriol63(4):299-304 (19847)] it is well observed that the novel organism ofthis invention has a production amount twice higher.

EXPERIMENTAL EXAMPLE 1

Measurement of Molecular Weight for Novel Phytase

2 l of novel strain solution, cultivated according to the procedure asdescribed in said Example, was centrifuged for 15 minutes. Then, thesupernatant solution was saturated with 50% acetone to precipitateproteins and the crude enzyme solution passed through dialysis membranewas purified on a column containing phenyl sepharose CL-4B, Resource Sand superose 12 HR 10/30 (all of which are manufactured by Pharmacia,Sweden) so as to isolate the phytase enzyme only. The analytical resultsby said column are represented in the attached drawing FIG. 2. Phytaseenzyme was again on SDS-PAGE electrophoresis and the results show thatthe molecular weight of phytase produced by a novel strain is 43,000dalton and isoelectric point is 5.6.

Further, the enzyme protein, so isolated, was used to determine thesequence of N-terminal amino acid using Protein/peptide Sequencer(Applied Biosystems, USA) and its results are represented in thesequence listing.

SEQ ID NO: 1 is the sequence of N-terminal amino acids of the phytaseproduced from a novel strain of this invention; SEQ ID NO: 2 is thesequence of the N-terminal amino acids of the phytase produced from E.coli [Greiner, et al., Purification and Characterization of Two Phytasesfrom Escherichia coli, Arch Biochem Biophys 303:107(1993)]; SEQ ID NO: 3is the sequence of the N-terminal amino acids of the phytase producedfrom Aspegillus ficuum [Ullah, A. H., Aspergillus Ficuum Phytase:Partial Primary Structure, Substrate Selectivity, and KineticCharacterization, Prep Biochem 18:459-72 (1988)].

From the results of the sequence listing, it is revealed that thephytase enzyme produced by Bacillus sp. DS11 (KCTC 0231BP) of thisinvention is a novel enzyme.

EXPERIMENTAL EXAMPLE 2

Activity and Stability of Novel Phytase to Temperature and pH

The optimal temperature was proved to be 65° C. by using the enzymeisolated by the same method as described in the experimental example 2.To measure the thermal stability, the enzyme was left at eachtemperature for 10 minutes so as to assess the residual activity. Asshown in the attached drawing FIG. 3 (3-1), the results show that whenCa²⁺ was not added, the activity began to decrease at 40° C. but in caseof adding 5 mM calcium ion, the activity was stable up to 70° C. and 50%of the activity at 75° C. was sustained.

From the aforementioned results, a novel phytase of this invention maybe expected to a higher activity in the body of livestock. Therefore, itseems to be preferable that the feeds should be pelleted or extruded atmore than 75° C. so as to use them as processing ones.

The results of phytase activity in the different conditions of pH areshown in the attached drawing FIG. 3 (3-2) and the optimal pH was 7.0.Further, to measure the stability on pH, the enzyme was left at variousconditions of pH for 1 hour so as to assess the residual activity ofphytase. Even under the acidic condition of below pH 4, the enzyme wasproved to have higher enzymatic activity and from these results, it isjudged that the novel phytase of this invention may be extremely stableunder the acidic condition of stomach.

From the test results of the aforementioned temperatures and pH, it isnoted that the novel phytase of this invention may be applicable as afeed additive of monogastric animals.

EXPERIMENTAL EXAMPLE 3

Influence of Metal Ion and Inhibitor on Enzyme Activity

Influence of metal ion and inhibitor on enzyme activity is representedin the following table 3.

The results of table 3 show that the addition of 1 mM EDTA inhibited theentire enzyme activity and when Cu²⁺, Zn²⁺ and Mg²⁺ were added at theconcentration of 5 mM, about 50% of enzyme activity was reduced.

TABLE 3 Concentration Additives 1 mM 5 mM Non-addition 100 100 CuCl₂ 6343 ZnCl₂ 87 47 MgCl₂ 95 49 MnCl₂ 65 20 LiCl₂ 95 100 HgCl₂ 83 62 CaCl₂ 99116 RbCl₂ 103 102 EDTA 7.5 7.6 PMSF 86 88

EXPERIMENTAL EXAMPLE 4

Influence of Novel Phytase Added to Feed for Broiler on EnvironmentalContamination

To estimate the influence relation of availability and released amountsof phosphate when phytase is added, broilers were divided into threegroups such as novel phytase group, soybean-extracted plant phytasegroup and mold phytase group on the market (manufacturer: Sigma). Eachof hatched-out 200 males of Avaachre broiler chicken was announcedpublicly for this experiment. The cultured medium containing phytase wasultrafiltrated and further concentrated at low temperature under vacuumand dried by lyophilizer. Each 500 unit of this lyophilized phytase wasadded to per kg of feed. The same amount of soybean phytase or moldphytase as above was also added to the feeds and the results wererepresented in the following table 4.

TABLE 4 Plant (soybean) Commercial Novel Classification phytase moldphytase phytase Phosphate contents in feeds (g/kg) 5.5 5.5 5.5 Intake oftotal feeds (g/kg) 2.7 2.7 2.7 Intake of phosphate (g/head) 15.1 15.215.1 Accumulation of phosphate (g/head) 7.1 7.8 8.8 Discharge ofphosphate (g/head) 8.0 7.4 6.3 Absorption of phosphate (%) 47 51 58

The results of Table 4 revealed that when novel phytase was fed tomonogastric animals, the phosphate contents released were lower than thesoybean group or mold phytase group, since the phosphate availability inthe former was higher than that of the latters; Namely, the activity ofnovel phytase was superior under acid-fast and acidic conditions andphytictaine within the grains was degraded in the intestine of animalsby the novel phytase in more effective manner.

[Sequence table]

Sequencing No.: 1

Sequencing length: 15

Sequencing form: amino acid

Shape: straight chain

Sequencing type: protein

Sequence: Ser-Asp-Pro-Tyr-His-Phe-Thr-Val-Asn-Ala-Ala-Xaa-Glu-Thr-Glu

Sequencing No.: 2

Sequencing length: 11

Sequencing form: amino acid

Shape: straight chain

Sequencing type: protein

Sequence: Ser-Glu-Pro-Glu-Leu-Lys-Leu-Glu-Ala-Val-Val

Sequencing No.: 3

Sequencing length: 12

Sequencing form: amino acid

Shape: straight chain

Sequencing type: protein

Sequence: Pro-Ala-Ser-Arg-x-Gin-Ser-Ser-Cys-Asp-Thr-Val

3 1 15 PRT Bacillus sp. DS 11 Xaa = any amino acid 1 Ser Asp Pro Tyr HisPhe Thr Val Asn Ala Ala Xaa Glu Thr Glu 1 5 10 15 2 11 PRT Escherichiacoli 2 Ser Glu Pro Glu Leu Lys Leu Glu Ala Val Val 1 5 10 3 12 PRTAspegillus ficuum Xaa = any amino acid 3 Pro Ala Ser Arg Xaa Gln Ser SerCys Asp Thr Val 1 5 10

What is claimed is:
 1. A biologically pure culture of Strain Bacillussp. DS11 (KCTC 0231BP).
 2. An isolated phytase produced by the Bacillussp. of claim 1 wherein the phytase has all of the following identifyingcharacteristics: Optimal temperature 65° C. Optimal pH 7.0 MolecularWeight 43,000 daltons Isoelectric point 5.6 Sequence of N-terminal aminoacid SEQ ID NO:
 1. 3. The method of using the isolated phytase, producedby strain Bacillus sp. DS11 (KCTC 0231BP), of claim 2 as a feed additivecomprising: a. preparing the phytase and b. adding it to feed.